Perturbation of the T cell receptor (TCR)/CD3 complex by antigen in the context of the appropriate MHC or by anti-receptor antibodies (Ab) initiates a cascade of events which include protein tyrosine kinase activation, the phosphorylation of an inositol phospholipid (InsPL)-specific phospholipase C (PLC) isozyme, PLCgamma-1, activation of InsPL hydrolysis, and the generation of second messengers that control protein kinase C activation and Ca2+ mobilization. PLCgamma-1 contains a number of independent sub-domains, including src-homology (SH) and plekstrin-homology (PH) domains. These domains function as docking sites in protein/protein interactions and are likely to play a critical role in coupling PLCgamma-1 to the TCR/CD3 complex and in regulating its activity. Recombinant glutathione S-transferase fusion proteins encompassing individual or multiple PLCgamma-1 sub-domains were used to screen for binding proteins. A number of phosphoproteins interacting specifically with individual SH domains were detected. A phosphoproteins of 120 kDa binding to the SH3 domain was identified as the protein product of the c-cbl proto-oncogene, a protein of unknown function, but involved in early activation events in response to TCR signaling, as indicated by its rapid phosphorylation by tyrosine kinases in response to mitogens or antibody-mediated ligation of the receptor. The interaction between the two native proteins was demonstrated by co-precipitating PLCgamma-1 with an anti-serum directed against p120c-cbl and blocking with an SH3-binding peptide. High level of expression of c-cbl in transiently transfected human Jurkat T leukemia cells have been obtained and will be used a as model to assess p120c-cbl function. In addition, transient expression in Jurkat cells of PLCgamma-1 SH subdomains as independent proteins has been obtained and will be used in conjunction with the expression of PLCgamma-1 SH mutants to asses their role in protein/protein interactions in vivo and T cell activation.